• 대한치과교정학회지
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• 제39권4호
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• pp.248-256
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• 2009

이 연구의 목적은 배양된 사람 치주인대 세포에서 파골세포의 형성에 관련된 물질을 합성, 분비할 수 있는지를 알아보고 압박력이 M-CSF, IL-$1{\beta}$, RANKL 및 OPG mRNA의 발현에 미치는 영향을 알아보고자 하였다. 교정치료를 목적으로 발치된 소구치에서 얻은 치주인대세포를 배양한 후 다양한 크기(0.5, 1.0, 2.0, 3.0, $4.0\;g/cm^2$)의 기계적 자극을 다양한 기간(0.5, 1.5, 6, 24, 48 hours) 동안 적용하고, M-CSF, IL-$1{\beta}$, RANKL, OPG mRNA 발현양의 변화를 검사하였다. 각각의 실험군에서 얻어진 mRNA에 대해 역전사 중합효소 연쇄반응검사를 시행하였다. 검사 결과 압박력은 사람 치주인대 세포에서 M-CSF mRNA를 발현시켰으며 M-CSF, IL-$1{\beta}$, RANKL mRNA의 발현양은 자극의 크기와 기간에 따라 증가하였다. 그러나 압박력은 사람 치주인대 세포에서 OPG mRNA의 발현양에 영향을 미치지 않는 것으로 나타났다. 이상의 결과는 기계적 자극이 치주인대 세포에서 M-CSF, IL-$1{\beta}$, RANKL mRNA의 발현양을 조절함으로 파골세포의 분화에 영향을 미칠 수 있음을 시사한다.

• Journal of Periodontal and Implant Science
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• 제36권2호
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• pp.335-344
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• 2006

Osteoblast or bone marrow stromal cell-derived RANKL is the major effector molecule essential for osteoclastogenesis. Previous studies have shown that tetracyclines have beneficial therapeutic effects in the prevention and treatment of inflammatory bone disease including periodontal disease. Periodontal ligament cells are thought not only to play an important role in the progression of periodontal disease, but to play an important role in alveolar bone remodeling. Previous studies indicated that receptor activation of nuclear factor $\kappa\;B$ ligand (RANKL) and osteoprotegerin (OPG) are expressed in periodontal ligament cells by pro-inflammatory cytokine, such as $IL-1{\beta}$ and $TNF-{\alpha}$. This study was designed to investigate the inhibitory effect of doxycycline on RANKL and OPG mRNA in rat periodontal ligament cells induced by $IL-1{\beta}$ (1 ng/ml). The results are as follows; 1. MTT assay showed that doxycycline at the concentration of $1-50\;{\mu}g/m{\ell}$ didn't result in statistically significant cell death at day 1 and 3. 2. RANKL mRNA expression was increased to 2.6 folds by $IL-1{\beta}$. When cells were treated with doxycycline ($50{\mu}g/m{\ell}$), $IL-1{\beta}$ -induced mRANKL expression was reduced by 33%. In contrast to RANKL, OPG mRNA expression was not inhibited by pre-treatment with doxycycline. These results suggest that doxycycline decrease the expression of mRANKL resulting in regulation of osteoclastogenesisp in rat periodontal ligament cells.

• 운동영양학회지
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• 제23권1호
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• pp.13-20
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• 2019

[Purpose] The OPG/RANK/RANKL signaling is a new family of bone metabolism biomarkers belonging to the immune system. However, the bone metabolism response to long-term exercise in the RANKL/RANK/OPG signaling is less evident. The purpose of this study was to examine these biomarkers in healthy college females after 12-weeks combined exercise intervention. [Methods] Participants (N=22, 22.4±1.3yrs) were randomly divided in two different group: 12 in the control group and 10 in the exercise group performing combined exercise program that interventions was conducted 3 times per week for 12 weeks. The outcome measures included serum concentrations of RANKL, OPG and bone metabolic cytokines such as TNF-α and IL-6, and mRNA expressions of same variables from PBMC. VO_2max and bone mineral density (BMD) were measured at before and after exercise intervention. [Results] There were no significant differences in the serum RANKL, OPG concentrations and all RANKL/RANK/OPG signaling mRNA expression on interaction effect between group and time (NS). Also no significant differences were found in the serum TNF-α and IL-6 concentrations and mRNA expression (NS). The IL-6 mRNA expression only showed significant difference in the main effect of groups (p<.05). There were also no significant differences in the VO_2max and BMD on interaction effect between group and time (NS). [Conclusion] These results suggested that there were no effects on bone mineral density and RANKL/RANK/OPG signaling without the effect of 8-weeks combined exercise on cardiovascular endurance fitness.

• 생명과학회지
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• 제25권2호
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• pp.223-230
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• 2015

RCAN1은 calcineurin을 억제하는 내인성 단백질로 calcineurin-NFATc1 신호전달 경로와 관련된 질환의 병인에 중요한 역할을 담당한다. 특히 RCAN1-4 아형 유전자의 경우 NFATc1 전사인자에 의해 조절된다. RANKL 자극은 calcineurin-NFATc1 경로로 파골세포 분화를 유도하는데, RCAN1과 파골세포의 분화에 관련된 연구는 보고 된 바 없다. 따라서 본 연구는 RANKL 처리에 의해 파골세포 분화가 유도될 때 RCAN1이 calcineurin-NFATc1 경로에 미치는 영향을 in vitro에서 조사했다. 마우스로부터 분리한 골수단핵세포에 RANKL을 처리하여 파골세포 분화를 유도했다. RANKL 처리 후 조사 대상 유전자의 mRNA 발현과 단백질 발현을 각각 RT-PCR과 Western blot로써 측정했다. 마우스 RCAN1-4 vector를 파골전구세포인 RAW 264.7 단핵세포주와 골수단핵세포에 형질도입(transfection)시켜 RCAN1-4 유전자의 과발현을 유도했다. 형질도입 후 파골세포 분화의 형태적 변화는 TRAP 염색을 통해 관찰했다. RANKL 처리 후 NFATc1, calcineurin, RCAN1-4 mRNA 발현은 크게 증가했다. 단백질 발현의 경우 NFATc1과 RCAN1은 증가했으나 calcineurin은 대조군과 차이가 없었다. RCAN1-4 유전자의 과발현 유도 시 RCAN1-4 mRNA는 크게 증가되었으나 RCAN1 단백질 발현은 증가되지 않았다. 특히 RANKL 존재 시 RCAN1 유전자를 knock-down시켜도 RCAN1 발현은 정상적으로 유지되었다. 한편, NFATc1 발현은 과발현 유도시 감소했고 knock-down 유도 시 증가하는 경향을 보였다. RCAN1-4 유전자 과발현을 유도한 골수단핵세포에서 배양 5일 후 파골세포 분화는 대조군과 차이가 없었다. 이러한 결과는 RANKL에 의한 파골세포 분화 시 RCAN1이 calcineurin-NFATc1 경로를 통해 파골세포 분화에 미치는 영향은 제한적일 것으로 사료된다.

• International Journal of Oral Biology
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• 제30권1호
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• pp.23-30
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• 2005

Bone remodeling is a process controlled by the action of two major bone cells; the bone forming osteoblast and the bone resorbing osteoclast. In the process of osteoclastogenesis, stromal cells and osteoblast produce RANKL, OPG, and M-CSF, which in turn regulate the osteoclastogenesis. During the bone resorption by activated osteoclasts, extracellular $Ca^{2+}/{PO_4}^{2-}$ concentration and degraded organic materials goes up, providing the hypertonic microenvironment. In this study, we tested the effects of hypertonicity due to the degraded organic materials on osteoclastogenesis in co-culture system. It was examined the cellular response of osteoblastic cell in terms of osteoclastogenesis by applying the sucrose, and mannitol, as a substitute of degraded organic materials to co-culture system. Apart from the sucrose, mannitol, and NaCl was tested to be compared to the effect of organic osmotic particles. The addition of sucrose and mannitol (25, 50, 100, 150, or 200 mM) to co-culture medium inhibited the number of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells induced by 10 nM $1{\alpha},25(OH)_2vitaminD_3$ ($1{\alpha},25(OH)_2D_3$). However, NaCl did exert harmful effect upon the cells in this co-culture system, which is attributed to DNA damage in high concentration of NaCl. To further investigate the mechanism by which hypertonicity inhibits $1{\alpha},25(OH)_2D_3$-induced osteoclastogenesis, the mRNA expressions of receptor activator of nuclear factor (NF)-kB ligand (RANKL) and osteoprotegerin (OPG) were monitored by RT-PCR. In the presence of sucrose (50 mM), RANKL mRNA expression was decreased in a dose-dependent manner, while the change in OPG and M-CSF mRNA were not occurred in significantly. The RANKL mRNA expression was inhibited for 48 hours in the presence of sucrose (50 mM), but such a decrement recovered after 72 hours. However, there were no considerable changes in the expression of OPG and M-CSF mRNA. Conclusively, these findings strongly suggest that hypertonic stress down-regulates $1{\alpha},25(OH)_2D_3$-induced osteoclastogenesis via RANKL signal pathway in osteoblastic cell, and may playa pivotal role as a regulator that modulates osteoclastogenesis.

• International Journal of Oral Biology
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• 제38권2호
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• pp.67-72
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• 2013

This study examined the anti-osteoclastogenic effects of baicalin on receptor activator of NF-${\kappa}$B ligand (RANKL)-induced RAW264.7 cells. Baicalin is a flavonoid that is produced by Scutellaria baicalensis and is known to have multiple biological properties, including antibacterial, anti-inflammatory and analgesic effects. The effects of baicalin on osteoclasts were examined by measuring 1) cell viability; 2) the formation of tartrate-resistant acid phosphatase (TRAP) (+) multinucleated cells; 3) RANK/RANKL signaling pathways and 4) mRNA levels of osteoclast-associated genes. Baicalin inhibited the formation of RANKL-stimulated TRAP (+) multinucleated cells and also suppressed the RANKL-stimulated activation of p-38, ERK, cSrc and AKT signaling. Baicalin also inhibited the RANKL-stimulated degradation of $I{\kappa}B$ in RAW264.7 cells. In addition, the RANKL-stimulated induction of NFATc1 transcription factors was found to be abrogated by this flavonoid. Baicalin was further found to decrease the mRNA expression of osteoclast-associated genes, including carbonic anhydrase II, TRAP and cathepsin K in the RAW264.7 cells. Our data thus demonstrate that baicalin inhibits osteoclastogenesis by inhibiting the RANKL-induced activation of signaling molecules and transcription factors in osteoclast precursors.

• 대한치과교정학회지
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• 제35권4호
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• pp.262-274
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• 2005

본 연구는 치주인대 세포에 지속적이고 점진적 인장력을 가하여 치아 이동 시 형성되는 인장부위의 기계적 자극에 대한 생화학적 전달과 치조골 흡수와 생성 조절 기전을 이해하고자 하였다 치주인대 세포가 배양된 유연한 성장 표면을 가진 배지에 지속적이고 점진적인 인장력을 가하고 골흡수 인자인 $PGE_2$와 골형성 인자인 ALP의 생성량을 1 3 5. 12시간 후에 측정하여 정량비교하였고 파골세포 분화기전을 조절하는 OPG RANKL의 인자들과 matrix metalloproteinase(MMP)-1, -8, -9, -13, tissue inhibitor of matrix metalloproteinase(TIMP)-1의 인자들을 역전사 중합효소 연쇄반응 검사하여 m-RNA 발현을 비교한 결과 치주인대 세포에 인장력을 가한 경우 대조 군보다 $PGE_2$의 농도가 적었고 (p<0.05) ALP의 농도 변화는 없었으며 OPG의 mRNA 발현이 증가하였으나, RANKL의 mRNA 발현은 감소하였다 그리고 TIMP-1과 MMP-1 -8 -9, -13의 mRNA 발현이 대조군과 차이가 없었다. 이상의 연구에서 사람의 치주인대 세포는 점진적이고 지속적인 인장력에 대한 반응으로 $PGE_2$의 생성과 RANKL의 mRNA 발현은 감소하고 OPG의 mRNA 발현은 증가하여 골흡수를 억제하는 효과를 보이는 것으로 나타났다.

• International Journal of Oral Biology
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• 제42권4호
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• pp.163-168
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• 2017

Osteoporosis is a metabolic bone disease that is characterized by low bone mass resulting from an increase in bone resorption relative to bone formation. The most current therapies for osteoporosis have focused on inhibiting bone resorption by osteoclasts. The purpose of this study is to develop new anabolic agents for treatment of osteoporosis that have fewer risks compared to conventional therapies. We searched the natural products that were derived from the traditional Asian medicines which have been used for treatment of bone related diseases. Icaritin is a flavonoid glycoside derived from the herb Epimedium which has beneficial effects on bone formation. To determine the effect of icaritin on bone formation, we examined the effect of icaritin on MC3T3-E1 cell proliferation and differentiation. For determining the effects of icaritin on proliferation, we performed the MTT assay using MC3T3-E1 cells. To evaluate whether icaritin could promote the osteogenic differentiation of MC3T3-E1 cells, alkaline phosphatase (ALP) activity and mRNA expressions of Runx2, osteocalcin (OCN), RANKL, and osteoprotegerin (OPG) were determined. Icaritin increased MC3T3-E1 cell proliferation. Icaritin increased the ALP activity of MC3T3-E1 cells on 72 hour culture in osteogenic media. mRNA expression of Runx2 was increased after 24 hour culture with icaritin. mRNA expression of osteocalcin was increased after 72 hour culture with icaritin. In addition, icaritin increased the mRNA expressions of OPG and RANKL. However, icaritin increased the mRNA expression of OPG much more than that of RANKL, and then, it increased the OPG/RANKL ratio. These results suggest that icaritin promotes osteogenic differentiation of osteoblasts and decreases osteoclast formation regulated by osteoblasts.

• The Korean Journal of Physiology and Pharmacology
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• 제19권2호
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• pp.111-118
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• 2015

Osteoprotegerin (OPG), receptor activator of NF-${\kappa}B$ ligand (RANKL)/receptor activator of NF-${\kappa}B$ (RANK) axis, and TNF-related apoptosis-inducing ligand (TRAIL) participate in vascular calcification process including atherosclerosis, but their contributions under high glucose (HG) and phosphate (HP) condition for a long-term period (more than 2 weeks) have not been fully determined. In this study, we evaluated the effects of HG and HP levels over 2 or 4 weeks on the progression of vascular calcification in rat vascular smooth muscle cells (VSMCs). Calcium deposition in VSMCs was increased in medium containing HG (30 mmol/L D-glucose) with ${\beta}$-glycerophosphate (${\beta}$-GP, 12 mmol/L) after 2 weeks and increased further after 4 weeks. OPG mRNA and protein expressions were unchanged in HG group with or without ${\beta}$-GP after 2 weeks. However, after 4 weeks, OPG mRNA and protein expressions were significantly lower in HG group with ${\beta}$-GP. No significant expression changes were observed in RANKL, RANK, or TRAIL during the experiment. After 4 weeks of treatment in HG group containing ${\beta}$-GP and rhBMP-7, an inhibitor of vascular calcification, OPG expressions were maintained. Furthermore, mRNA expression of alkaline phosphatase (ALP), a marker of vascular mineralization, was lower in the presence of rhBMP-7. These results suggest that low OPG levels after long term HG and phosphate stimulation might reduce the binding of OPG to RANKL and TRAIL, and these changes could increase osteo-inductive VSMC differentiation, especially vascular mineralization reflected by increased ALP activity during vascular calcification.

• The Korean Journal of Physiology and Pharmacology
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• 제17권1호
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• pp.65-71
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• 2013

The transient receptor potential melastatin type 7 (TRPM7) channel is a widely expressed non-selective cation channel with fusion to the C-terminal alpha kinase domain and regarded as a key regulator of whole body $Mg^{2+}$ homeostasis in mammals. However, the roles of TRPM7 during osteoclastogenesis in RAW264.7 cells and bone marrow-derived monocyte/macrophage precursor cells (BMMs) are not clear. In the present study, we investigate the roles of TRPM7 in osteoclastogenesis using methods of small interfering RNA (siRNA), RT-PCR, patch-clamp, and calcium imaging. RANKL (receptor activator of NF-${\kappa}B$ ligand) stimulation did not affect the TRPM7 expression and TRPM7-mediated current was activated in HEK293, RAW264.7, and BMM cells by the regulation of $Mg^{2+}$. Knock-down of TRPM7 by siTRPM7 reduced intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) increases by 0 mM $[Mg^{2+}]_e$ in HEK293 cells and inhibited the generation of RANKL-induced $Ca^{2+}$ oscillations in RAW264.7 cells. Finally, knock-down of TRPM7 suppressed RANKL-mediated osteoclastogenesis such as activation and translocation of NFATc1, formation of multinucleated cells, and the bone resorptive activity, sequentially. These results suggest that TRPM7 plays an essential role in the RANKL-induced $[Ca^{2+}]_i$ oscillations that triggers the late stages of osteoclastogenesis.